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. 2020 Feb 23;12(2):249.
doi: 10.3390/v12020249.

Chicken eEF1α Is a Critical Factor for the Polymerase Complex Activity of Very Virulent Infectious Bursal Disease Virus

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Free PMC article

Chicken eEF1α Is a Critical Factor for the Polymerase Complex Activity of Very Virulent Infectious Bursal Disease Virus

Bo Yang et al. Viruses. .
Free PMC article

Abstract

Infectious bursal disease (IBD) is an immunosuppressive, highly contagious, and lethal disease of young chickens caused by IBD virus (IBDV). It results in huge economic loss to the poultry industry worldwide. Infection caused by very virulent IBDV (vvIBDV) strains results in high mortality in young chicken flocks. However, the replication characteristics of vvIBDV are not well studied. Publications have shown that virus protein 3 (VP3) binds to VP1 and viral double-stranded RNA, and together they form a ribonucleoprotein complex that plays a key role in virus replication. In this study, vvIBDV VP3 was used to identify host proteins potentially involved in modulating vvIBDV replication. Chicken eukaryotic translation elongation factor 1α (cheEF1α) was chosen to further investigate effects on vvIBDV replication. By small interfering RNA-mediated cheEF1α knockdown, we demonstrated the possibility of significantly reducing viral polymerase activity, with a subsequent reduction in virus yields. Conversely, over-expression of cheEF1α significantly increased viral polymerase activity and virus replication. Further study confirmed that cheEF1α interacted only with vvIBDV VP3 but not with attenuated IBDV (aIBDV) VP3. Furthermore, the amino acids at the N- and C-termini were important in the interaction between vvIBDV VP3 and cheEF1α. Domain III was essential for interactions between cheEF1α and vvIBDV VP3. In summary, cheEF1α enhances vvIBDV replication by promoting the activity of virus polymerase. Our study indicates cheEF1α is a potential target for limiting vvIBDV infection.

Keywords: VP3; eEF1α; polymerase; vvIBDV.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Very virulent infectious bursal disease virus (vvIBDV) virus protein 3 (VP3) was used to fish potential associated host factors. (A) Six different gel bands were detected in vvIBDV VP3 immunoprecipitated cell lysates compared with pCAGGS vector control sample. DF-1 cells were transfected with eukaryotic expression plasmids harboring the HA-tagged vvIBDV VP3 or empty vector pCAGGS. Cells were lysed with commercial IP and WB lysis buffer after 36 h post-transfection, and cell lysates were incubated with anti-HA protein A/G at 4 °C overnight. Then, the immunoprecipitated protein A/G were collected for SDS-PAGE, and the different gel bands were sent for LC–MS/MS identification. Meanwhile, the expression of transfected proteins was confirmed by Western blotting with anti-HA antibody, and results were shown in (B).
Figure 2
Figure 2
Chicken eukaryotic translation elongation factor 1α (cheEF1α) promoted the replication of vvIBDV. (A) Effects of siRNAs on the cheEF1α expression. 293T cells were transfected with Flag-tagged cheEF1α accompanied with siRNAs (RNAi#1, RNAi#2, or RNAi#3) or scrambled siRNA. Cell lysates were harvested 48 h after transfection and tested by Western blotting with anti-Flag antibody. Endogenous β-actin expression was used as an internal control. (B) Yields of viral proteins in cheEF1α knockdown cells. Viral proteins loads were analyzed by Western blotting at 24 h, 48 h, and 72 h post-infection (p.i.) with vvIBDV Gx at multiplicity of infection (MOI) = 1 compared the potent cheEF1α siRNA treatment to scrambled siRNA treatment. The amount of cheEF1α indicated the efficiency of siRNA silencing and β-actin was used as the loading control. (C) Typical survival curve of chicken embryos infected with extracellular progeny virus in cheEF1α knockdown cells. Progeny viruses in extracellular of cells with or without cheEF1α knockdown were harvested at 72 h p.i., and chicken embryos were infected with 10-fold diluted progeny viruses, with the number of deaths for 9 days post-infection being recorded. One of three independent tests was shown as survival curve, and these three independent tests of 50% embryo lethal dose (ELD50) were summarized in (D). (E) Yields of viral proteins in cheEF1α over-expression cells. Viral protein loads were analyzed by Western blotting at 48 h and 72 h p.i. with vvIBDV Gx at MOI = 1 with or without cheEF1α over-expression. (F) Typical survival curve of chicken embryos infected with extracellular progeny virus in cheEF1α over-expression cells. The progeny virus titers based on 50% embryo lethal dose (ELD50) are summarized in (G). Error bars and mean ± SD were calculated on the basis of three independent experiments. The statistical analysis of survival curve was analyzed with GraphPad Prism 6 software, and the other statistical analysis was performed with t-tests considered significant with one asterisk as p < 0.05 and two asterisks as p < 0.01.
Figure 3
Figure 3
cheEF1α interacts with vvIBDV VP3, but not aIBDV. VP3 (A) 293T cells were transfected with HA-vvIBDV VP3 and Flag-cheEF1α expression plasmids. Cell lysates were prepared at 36 h post transfection and immunoprecipitated with anti-HA antibody and immunoblotted with anti-Flag or anti-HA antibodies. (B) 293T cells were transfected as in (A). Cell lysates were prepared at 36 hours post transfection and immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Flag or anti-HA antibodies. (C) 293T cells were transfected with HA-vvIBDV VP3 and Flag-cheEF1α or HA-aIBDV VP3 and Flag-cheEF1α expression plasmids. Cell lysates were prepared at 36 h post transfection and immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Flag or anti-HA antibodies. (D) 293T cells were transfected with HA-vvIBDV VP3 and/or Flag-cheEF1α for 36 h and then fixed and processed for dual-immunostaining. Cell nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI) (blue). VP3 (red) and cheEF1α (green) proteins were visualized by labelling with anti-HA and/or anti-Flag antibodies, respectively, and were observed using confocal microscopy. Scale bars were marked down right corner. VP3 staining is shown in red, and cheEF1α staining is shown in green; merged images are shown with areas of co-localization in yellow. Each co-immunoprecipitation experiment was repeated three times.
Figure 4
Figure 4
Both N- and C-terminal amino acids of VP3 are essential in the interaction between vvIBDV VP3 and cheEF1α. (A) Four mutant plasmids with only one amino acid mutation including vvIBDV-VP3-H28Q, -VP3-P226L, -VP3-V235A, and -VP3-A250T were constructed using vvIBDV VP3 as the template. (B) Four mutant plasmids aIBDV-VP3-Q28H, -VP3-L226P, -VP3-A235V, and -VP3-T250A with one amino acid substitution were constructed with attenuated VP3 as the template. (C) 293T cells were transfected expression plasmids with Flag-cheEF1α and HA-vvIBDV VP3, Flag-cheEF1α and vvIBDV-VP3-H28Q, Flag-cheEF1α and vvIBDV-VP3-P226L, Flag-cheEF1α and vvIBDV-VP3-V235A, or Flag-cheEF1α and vvIBDV-VP3-A250T. Cell lysates were prepared at 36 h post-transfection and immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Flag or anti-HA antibodies. (D) 293T cells were transfected expression plasmids with Flag-cheEF1α and HA-aIBDV VP3, Flag-cheEF1α and aIBDV-VP3-Q28H, Flag-cheEF1α and aIBDV-VP3-L226P, Flag-cheEF1α and aIBDV-VP3-A235V, or Flag-cheEF1α and aIBDV-VP3-T250A. Cell lysates were prepared at 36 h post-transfection and immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Flag or anti-HA antibodies. Each co-immunoprecipitation experiment was repeated three times.
Figure 5
Figure 5
The third domain of cheEF1α interacted with vvIBDV VP3. (A) The expression plasmids harboring different domains of cheEF1α were depicted as cheEF1α-DI, cheEF1α-DII, cheEF1α-DIII, cheEF1α-DI+II, and cheEF1α-DII+III. (B) 293T cells were transfected expression plasmids with HA-vvIBDV VP3 and Flag-cheEF1α, HA-vvIBDV VP3 and Flag-cheEF1α-DI, HA-vvIBDV VP3 and Flag-cheEF1α-DII, HA-vvIBDV VP3 and Flag-cheEF1α-DIII, HA-vvIBDV VP3 and Flag-cheEF1α-DI+II, or HA-vvIBDV VP3 and Flag-cheEF1α-DII+III. Cell lysates were prepared at 36 h post transfection and immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Flag or anti-HA antibodies. Each co-immunoprecipitation experiment was repeated three times.
Figure 6
Figure 6
cheEF1α enhances the activity of vvIBDV polymerase. (A,B) Effects of cheEF1α over-expression on virus polymerase activity. The polymerase activity assays were performed in 293T cells expressing vvIBDV segment B-driven luciferase minigenome system, accompanied with VP3, segment B, TK, and cheEF1α or empty vector pCAGGS. VP1, VP3, and cheEF1α protein expressions were analyzed by Western blotting with β-actin as a loading control. (C,D) Effects of eEF1α knockdown on virus polymerase activity. 293T cells were first transfected with commercial competent eEF1α siRNAs or scrambled control siRNAs. Polymerase activity assays were performed at 36 h post-second transfection with viral polymerase system as described in (A). VP1, VP3, and eEF1α protein expressions were analyzed by Western blotting with β-actin as a loading control. Error bars, mean ± SD were calculated on the basis of three independent experiments. Statistical analysis was performed with the t-test with significance shown by two asterisks as p < 0.01.

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